In the production of specific proteins in a recombinant host by recombinant DNA technology, there are many advantages to having the host express and secrete the desired protein. That is, when a desired protein is expressed directly within the host cell, if there is any toxicity which inhibits growth or compromises the survival of the host cell, this toxicity can be avoided by the secretion of the protein. Even when there is no toxicity, as the protein accumulates in the host cell, it may inhibit the host cell growth. This, too, can be avoided by secretory expression. In addition, systems which accumulate protein in the host cell may also denature it, rendering it insoluble. This problem also can be avoided by secretory expression. Moreover, when commercially producing protein by recombinant DNA technology in a system which accumulates the desired protein intracellularly, it is necessary to destroy the cell in order to refine the protein, and it must be purified from the debris of the cellular destruction. This makes it difficult to obtain a protein of high purity. On the other hand, when producing a protein by a secretory expression system the protein only must be harvested from the culture broth, minimizing the problem of separating impurities derived from the recombinant host. This is a great advantage. Finally, most protein undergoes some modification, such as the addition of a sugar moiety, the formation of a disulfide bond, activation by limited hydrolysis of the inert proprotein, phosphorylation of specific amino acids, or carboxylation before activation. Some of these functions are performed by the themselves, and several of these modifications take place in the process of secretion. Therefore, a system which produces protein by secretory expression, as compared to a system which accumulates protein intracellularly, may be expected to generate proteins having a structure and function much close to the native protein.
Some things are known about the properties of the signal peptide, and the characteristics of its amino acid sequence seem to be as follows. There are many basic amino acids near the N-terminal, and there are many polar amino acids near the portion which is digested by signal peptidase on the C-terminal side, while a sequence hydrophobic amino acids fill in the space between these two areas. The basic amino acids near the N-terminal interact with the phospholipids on the internal surface of the cell membrane, and the sequence of hydrophobic amino acids in the middle region playes an important role in passing the protein through the cell membrane. The polar amino acids at the C-terminal are believed to play some role in recognition during digestion by signal peptidase. These characteristics are extremely similar from procaryotes to higher animals, suggesting a common mechanism for protein secretion. (M. S. Briggs and L. M. Gierasch, Adv. Protein Chem., 38, 109-180 (1986); G. yon Heijne, EMBO J., 3, 2315-2318 (1984)).
Human serum albumin is encoded on the gene as a prepro type protein (see Japanese Patent Application (OPI) No. 29985/87 (the term OPI used herein means an unexamined published application.) or EP-A-206733; A. Dugaiczyk et al. Proc. Natl. Acad. Sci. USA, 79, 71-75 (1982)). The DNA and amino acid sequence in the vicinity of the N-terminal of mature human serum albumin beginning from the signal peptide essential for secretion are shown in Table 1 below.
TABLE 1 __________________________________________________________________________ ##STR1## __________________________________________________________________________
The signal peptide, composed of 18 amino acid is removed at the time of secretion. The propeptide, composed of 6 amino acids, is removed by processing, and mature human serum albumin, composed of 585 amino acids, and having an N-terminal amino acid sequence of Asp-Ala-His-Lys-Ser . . . , is obtained.
Since yeast secrete less extracellular proteases and are capable of adding sugar moieties to its secreta, yeast is excellent for the secretory expression of foreign proteins.
Several cases of signal peptides which contributes to the secretory expression in cells other than yeast, but which also function in yeast, have been reported. Examples include the secretory expression in yeast of human lysozyme using the chicken lysozyme signal peptide (Jigami, BIOINDUSTRY, 4, 117-123 (1987)), secretory expression in yeast of thaumatin using the signal peptide for plant protein thaumatin (L. Edens, I. Bom, A. M. Ledeboer, J. Maat, M. Y. Toonen, C. Visser and C. T. Verrips, Cell, 37, 629-633 (1984)), and secretory expression in yeast of human interferon using the signal peptide for human interferon-.alpha. (R. A. Hitzeman, D. W. Leung, L. J. Perry, W. J. Kohr, H. L. Levine and D. V. Doeddel, Science, 219, 620-625 (1983)).
The truth is, however, that the signal peptide contributing to secretory expression in cells other than yeast does not always function in yeast.